Glycosylation Pathways in Humans and Lower Eukaryotes
After DNA is transcribed and translated into a protein, further post-translational processing involves the attachment of sugar residues, a process known as glycosylation. Different organisms produce different glycosylation enzymes (glycosyltransferases and glycosidases), and have different substrates (nucleotide sugars) available, so that the glycosylation patterns as well as composition of the individual oligosaccharides, even of the same protein, will be different depending on the host system in which the particular protein is being expressed. Bacteria typically do not glycosylate proteins, and if so only in a very unspecific manner (Moens and Vanderleyden (1997) Arch Microbiol. 168(3):169-175). Lower eukaryotes such as filamentous fungi and yeast add primarily mannose and mannosylphosphate sugars. The resulting glycan is known as a “high-mannose” type glycan or a mannan. Plant cells and insect cells (such as Sf9 cells) glycosylate proteins in yet another way. By contrast, in higher eukaryotes such as humans, the nascent oligosaccharide side chain may be trimmed to remove several mannose residues and elongated with additional sugar residues that typically are not found in the N-glycans of lower eukaryotes. See, e.g., Bretthauer, et al. (1999) Biotechnology and Applied Biochemistry 30:193-200; Martinet, et al. (1998) Biotechnology Letters 20:1171-1177; Weikert, et al. (1999) Nature Biotechnology, 17:1116-1121; M. Malissard, et al. (2000) Biochemical and Biophysical Research Communications 267:169-173; Jarvis, et al., (1998) Current Opinion in Biotechnology 9:528-533; and Takeuchi (1997) Trends in Glycoscience and Glycotechnology 9:S29-S35.
Synthesis of a mammalian-type oligosaccharide structure begins with a set of sequential reactions in the course of which sugar residues are added and removed while the protein moves along the secretory pathway in the host organism. The enzymes which reside along the glycosylation pathway of the host organism or cell determine the resulting glycosylation patterns of secreted proteins. Thus, the resulting glycosylation pattern of proteins expressed in lower eukaryotic host cells differs substantially from the glycosylation pattern of proteins expressed in higher eukaryotes such as humans and other mammals (Bretthauer, 1999). The structure of a typical fungal N-glycan is shown in FIG. 1A.
The early steps of human glycosylation can be divided into at least two different phases: (i) lipid-linked Glc3Man9GlcNAc2 oligosaccharides are assembled by a sequential set of reactions at the membrane of the endoplasmic reticulum (ER) (FIG. 13) and (ii) the transfer of this oligosaccharide from the lipid anchor dolichyl pyrophosphate onto de novo synthesized protein. The site of the specific transfer is defined by an asparagine (Asn) residue in the sequence Asn-Xaa-Ser/Thr (SEQ ID NOs:1 and 2) where Xaa can be any amino acid except proline (Gavel and von Heijne (1990) Protein Eng. 3:433-42). Further processing by glucosidases and mannosidases occurs in the ER before the nascent glycoprotein is transferred to the early Golgi apparatus, where additional mannose residues are removed by Golgi specific alpha (α)-1,2-mannosidases. Processing continues as the protein proceeds through the Golgi. In the medial Golgi, a number of modifying enzymes, including N-acetylglucosaminyl transferases (GnTI, GnTII, GnTIII, GnTIV and GnTV), mannosidase II and fucosyltransferases, add and remove specific sugar residues. Finally, in the trans-Golgi, galactosyltranferases (GalT) and sialyltransferases (ST) produce a glycoprotein structure that is released from the Golgi. It is this structure, characterized by bi-, tri- and tetra-antennary structures, containing galactose, fucose, N-acetylglucosamine and a high degree of terminal sialic acid, that gives glycoproteins their human characteristics. The structure of a typical human N-glycan is shown in FIG. 1B. See also FIGS. 14 and 15 for steps involved in mammalian-type N-glycan processing.
In all eukaryotes studied to date, glycoproteins are derived from a common lipid-linked oligosaccharide precursor Glc3Man9GlcNAc2-dolichol-pyrophosphate. Within the endoplasmic reticulum, synthesis and processing of dolichol pyrophosphate bound oligosaccharides are identical between all known eukaryotes. However, further processing of the core oligosaccharide by fungal cells, e.g., yeast, differs significantly from humans as it moves along the secretory pathway.
In yeast, these steps are catalyzed by Golgi residing mannosyltransferases, like Och1p, Mnt1p and Mnn1p, which sequentially add mannose sugars to the core oligosaccharide. The resulting structure is undesirable for the production of human-like proteins and it is thus desirable to reduce or eliminate mannosyltransferase activity. Mutants of S. cerevisiae, deficient in mannosyltransferase activity (for example och1 or mnn9 mutants) have been shown to be non-lethal and display reduced mannose content in the oligosaccharide of yeast glycoproteins. Other oligosaccharide processing enzymes, such as mannosylphosphate transferases, may also have to be eliminated depending on the host's particular glycosylation pathways.
Sugar Nucleotide Precursors
The N-glycans of animal glycoproteins typically include galactose, fucose, and terminal sialic acid. These sugars are not found on glycoproteins produced in yeast and filamentous fungi. In humans, the full range of nucleotide sugar precursors (e.g., UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose, GDP-fucose, etc.) are synthesized in the cytosol and transported into the Golgi, where they are attached to the core oligosaccharide by glycosyltransferases. (Sommers and Hirschberg (1981) J. Cell Biol. 91(2):A406-A406; Sommers and Hirschberg (1982) J. Biol. Chem. 257(18):811-817; Perez and Hirschberg (1987) Methods in Enzymology 138:709-715).
Glycosyl transfer reactions typically yield a side product which is a nucleoside diphosphate or monophosphate. While monophosphates can be directly exported in exchange for nucleoside triphosphate sugars by an antiport mechanism, diphosphonucleosides (e.g., GDP) have to be cleaved by phosphatases (e.g. GDPase) to yield nucleoside monophosphates and inorganic phosphate prior to being exported. This reaction is important for efficient glycosylation; for example, GDPase from Saccharomyces cerevisiae (S. cerevisiae) has been found to be necessary for mannosylation. However that GDPase has 90% reduced activity toward UDP (Berninsone et al. (1994) J. Biol. Chem. 269(1):207-211). Lower eukaryotes typically lack UDP-specific diphosphatase activity in the Golgi since they do not utilize UDP-sugar precursors for Golgi-based glycoprotein synthesis. Schizosaccharomyces pombe, a yeast found to add galactose residues to cell wall polysaccharides (from UDP-galactose) has been found to have specific UDPase activity, indicating the potential requirement for such an enzyme (Berninsone et al. (1994) J. Biol. Chem. 269(1):207-211). UDP is known to be a potent inhibitor of glycosyltransferases and the removal of this glycosylation side product may be important to prevent glycosyl-transferase inhibition in the lumen of the Golgi (Khatara et al. (1974) Eur. J. Biochem. 44:537-560). See Berninsone et al. (1995) J. Biol. Chem. 270(24):14564-14567; Beaudet et al. (1998) Abc Transporters: Biochemical, Cellular, and Molecular Aspects 292: 397-413.
Sequential Processing of N-Glycans by Compartmentalized Enzyme Activities
Sugar transferases and glycosidases (e.g., mannosidases) line the inner (luminal) surface of the ER and Golgi apparatus and thereby provide a “catalytic” surface that allows for the sequential processing of glycoproteins as they proceed through the ER and Golgi network. The multiple compartments of the cis, medial, and trans Golgi and the trans-Golgi Network (TGN), provide the different localities in which the ordered sequence of glycosylation reactions can take place. As a glycoprotein proceeds from synthesis in the ER to full maturation in the late Golgi or TGN, it is sequentially exposed to different glucosidases, mannosidases and glycosyltransferases such that a specific carbohydrate structure may be synthesized. Much work has been dedicated to revealing the exact mechanism by which these enzymes are retained and anchored to their respective organelle. The evolving picture is complex but evidence suggests that stem region, membrane spanning region and cytoplasmic tail, individually or in concert, direct enzymes to the membrane of individual organelles and thereby localize the associated catalytic domain to that locus (see, e.g., Gleeson (1998) Histochem. Cell Biol. 109:517-532).
In some cases, these specific interactions were found to function across species. For example, the membrane spanning domain of α2,6-ST from rats, an enzyme known to localize in the trans-Golgi of the animal, was shown to also localize a reporter gene (invertase) in the yeast Golgi (Schwientek et al. (1995) J. Biol. Chem. 270(10):5483-9). However, the very same membrane spanning domain as part of a full-length α2,6-ST was retained in the ER and not further transported to the Golgi of yeast (Krezdorn et al. (1994) Eur. J. Biochem. 220(3):809-17). Full length GalT from humans was not even synthesized in yeast, despite demonstrably high transcription levels. In contrast, the transmembrane region of the same human GalT fused to an invertase reporter was able to direct localization to the yeast Golgi, albeit it at low levels. Schwientek and co-workers have shown that fusing 28 amino acids of a yeast mannosyltransferase (MNT1), a region containing a cytoplasmic tail, a transmembrane region and eight amino acids of the stem region, to the catalytic domain of human GalT are sufficient for Golgi localization of an active GalT. Other galactosyltransferases appear to rely on interactions with enzymes resident in particular organelles because, after removal of their transmembrane region, they are still able to localize properly.
Improper localization of a glycosylation enzyme may prevent proper functioning of the enzyme in the pathway. For example, Aspergillus nidulans, which has numerous α-1,2-mannosidases (Eades and Hintz (2000) Gene 255(1):25-34), does not add GlcNAc to Man5GlcNAc2 when transformed with the rabbit GnTI gene, despite a high overall level of GnTI activity (Kalsner et al. (1995) Glycoconj. J. 12(3):360-370). GnTI, although actively expressed, may be incorrectly localized such that the enzyme is not in contact with both of its substrates: UDP-GlcNAc and a productive Man5GlcNAc2 substrate (not all Man5GlcNAc2 structures are productive; see below). Alternatively, the host organism may not provide an adequate level of UDP-GlcNAc in the Golgi or the enzyme may be properly localized but nevertheless inactive in its new environment. In addition, Man5GlcNAc2 structures present in the host cell may differ in structure from Man5GlcNAc2 found in mammals. Maras and coworkers found that about one third of the N-glycans from cellobiohydrolase I (CBHI) obtained from T. reesei can be trimmed to Man5GlcNAc2 by A. saitoi 1,2 mannosidase in vitro. Fewer than 1% of those N-glycans, however, could serve as a productive substrate for GnTI. Maras et al. (1997) Eur. J. Biochem. 249:701-707. The mere presence of Man5GlcNAc2, therefore, does not assure that further in vivo processing of Man5GlcNAc2 can be achieved. It is formation of a productive, GnTI-reactive Man5GlcNAc2 structure that is required. Although Man5GlcNAc2 could be produced in the cell (about 27 mol %), only a small fraction could be converted to Man5GlcNAc2 (less than about 5%, see Chiba et al. WO 01/14522).
To date, there is no reliable way of predicting whether a particular heterologously expressed glycosyltransferase or mannosidase in a lower eukaryote will be (1), sufficiently translated (2), catalytically active or (3) located to the proper organelle within the secretory pathway. Because all three of these are necessary to affect glycosylation patterns in lower eukaryotes, a systematic scheme to achieve the desired catalytic function and proper retention of enzymes in the absence of predictive tools, which are currently not available, would be desirable.
Production of Therapeutic Glycoproteins
A significant number of proteins isolated from humans or animals are post-translationally modified, with glycosylation being one of the most significant modifications. An estimated 70% of all therapeutic proteins are glycosylated and thus currently rely on a production system (i.e., host cell) that is able to glycosylate in a manner similar to humans. Several studies have shown that glycosylation plays an important role in determining the (1) immunogenicity, (2) pharmacokinetic properties, (3) trafficking, and (4) efficacy of therapeutic proteins. It is thus not surprising that substantial efforts by the pharmaceutical industry have been directed at developing processes to obtain glycoproteins that are as “humanoid” or “human-like” as possible. To date, most glycoproteins are made in a mammalian host system. This may involve the genetic engineering of such mammalian cells to enhance the degree of sialylation (i.e., terminal addition of sialic acid) of proteins expressed by the cells, which is known to improve pharmacokinetic properties of such proteins. Alternatively, one may improve the degree of sialylation by in vitro addition of such sugars using known glycosyltransferases and their respective nucleotide sugars (e.g., 2,3-sialyltransferase and CMP-sialic acid).
While most higher eukaryotes carry out glycosylation reactions that are similar to those found in humans, recombinant human proteins expressed in the above mentioned host systems invariably differ from their “natural” human counterpart (Raju et al. (2000) Glycobiology 10(5): 477-486). Extensive development work has thus been directed at finding ways to improve the “human character” of proteins made in these expression systems. This includes the optimization of fermentation conditions and the genetic modification of protein expression hosts by introducing genes encoding enzymes involved in the formation of human-like glycoforms. Goochee et al. (1999) Biotechnology 9(12):1347-55; Andersen and Goochee (1994) Curr Opin Biotechnol. 5(5):546-49; Werner et al. (1998) Arzneimittelforschung. 48(8):870-80; Weikert et al. (1999) Nat Biotechnol. 17(11):1116-21; Yang and Butler (2000) Biotech. Bioeng. 68:370-80. Inherent problems associated with all mammalian expression systems have not been solved.
Glycoprotein Production Using Eukaryotic Microorganisms
Although the core oligosaccharide structure transferred to a protein in the endoplasmic reticulum is basically identical in mammals and lower eukaryotes, substantial differences have been found in the subsequent processing reactions which occur in the Golgi apparatus of fungi and mammals. In fact, even amongst different lower eukaryotes there exist a great variety of glycosylation structures. This has historically prevented the use of lower eukaryotes as hosts for the production of recombinant human glycoproteins despite otherwise notable advantages over mammalian expression systems.
Therapeutic glycoproteins produced in a microorganism host such as yeast utilizing the endogenous host glycosylation pathway differ structurally from those produced in mammalian cells and typically show greatly reduced therapeutic efficacy. Such glycoproteins are typically immunogenic in humans and show a reduced half-life (and thus bioactivity) in vivo after administration (Takeuchi (1997) Trends in Glycoscience and Glycotechnology 9:S29-S35). Specific receptors in humans and animals (i.e., macrophage mannose receptors) can recognize terminal mannose residues and promote the rapid clearance of the foreign glycoprotein from the bloodstream. Additional adverse effects may include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity.
Yeast and filamentous fungi have both been successfully used for the production of recombinant proteins, both intracellular and secreted (Cereghino and Cregg (2000) FEMS Microbiology Reviews 24(1):45-66; Harkki et al. (1989) Bio-Technology 7(6):596; Berka et al. (1992) Abstr. Papers Amer. Chem. Soc. 203:121-BIOT; Svetina et al. (2000) J. Biotechnol. 76(2-3):245-251). Various yeasts, such as K. lactis, Pichia pastoris, Pichia methanolica, and Hansenula polymorpha, have played particularly important roles as eukaryotic expression systems because they are able to grow to high cell densities and secrete large quantities of recombinant protein. Likewise, filamentous fungi, such as Aspergillus niger, Fusarium sp, Neurospora crassa and others, have been used to efficiently produce glycoproteins at the industrial scale. However, as noted above, glycoproteins expressed in any of these eukaryotic microorganisms differ substantially in N-glycan structure from those in animals. This has prevented the use of yeast or filamentous fungi as hosts for the production of many therapeutic glycoproteins.
Although glycosylation in yeast and fungi is very different than in humans, some common elements are shared. The first step, the transfer of the core oligosaccharide structure to the nascent protein, is highly conserved in all eukaryotes including yeast, fungi, plants and humans (compare FIGS. 1A and 1B). Subsequent processing of the core oligosaccharide, however, differs significantly in yeast and involves the addition of several mannose sugars. This step is catalyzed by mannosyltransferases residing in the Golgi (e.g., OCH1, MNT1, MNN1, etc.), which sequentially add mannose sugars to the core oligosaccharide. The resulting structure is undesirable for the production of humanoid proteins and it is thus desirable to reduce or eliminate mannosyltransferase activity. Mutants of S. cerevisiae deficient in mannosyltransferase activity (e.g., och1 or mnn9 mutants) have shown to be non-lethal and display a reduced mannose content in the oligosaccharide of yeast glycoproteins. Other oligosaccharide processing enzymes, such as mannosylphosphate transferase, may also have to be eliminated depending on the host's particular endogenous glycosylation pattern. After reducing undesired endogenous glycosylation reactions, the formation of complex N-glycans has to be engineered into the host system. This requires the stable expression of several enzymes and sugar-nucleotide transporters. Moreover, one has to localize these enzymes so that a sequential processing of the maturing glycosylation structure is ensured.
Several efforts have been made to modify the glycosylation pathways of eukaryotic microorganisms to provide glycoproteins more suitable for use as mammalian therapeutic agents. For example, several glycosyltransferases have been separately cloned and expressed in S. cerevisiae (GalT, GnTI), Aspergillus nidulans (GnTI) and other fungi (Yoshida et al. (1999) Glycobiology 9(1):53-8, Kalsner et al. (1995) Glycoconj. J. 12(3):360-370). However, N-glycans resembling those made in human cells were not obtained.
Yeasts produce a variety of mannosyltransferases (e.g., 1,3-mannosyltransferases such as MNN1 in S. cerevisiae; Graham and Emr (1991) J. Cell. Biol. 114(2):207-218), 1,2-mannosyltransferases (e.g., KTR/KRE family from S. cerevisiae), 1,6-mannosyltransferases (e.g., OCH1 from S. cerevisiae), mannosylphosphate transferases and their regulators (e.g. MNN4 and MNN6 from S. cerevisiae) and additional enzymes that are involved in endogenous glycosylation reactions. Many of these genes have been deleted individually giving rise to viable organisms having altered glycosylation profiles. Examples are shown in Table 1.
TABLE 1Examples of yeast strains having altered mannosylationStrainN-glycan (wild type)MutationN-glycan (mutant)ReferenceS. pombeMan>9GlcNAc2OCH1Man8GlcNAc2Yoko-o et al.(2001) FEBS Lett.489(1): 75-80S. cerevisiaeMan>9GlcNAc2OCH1/MNN1Man8GlcNAc2Nakanishi-Shindoet al. (1993)J.Biol. Chem.268(35): 26338-26345S. cerevisiaeMan>9GlcNAc2OCH1/MNN1/MNN4Man8GlcNAc2Chiba et al. (1998)J. Biol. Chem.273, 26298-26304P. pastorisHyperglycosylatedOCH1 (completeNotWelfide, Japanesedeletion)hyperglycosylatedApplicationPublication No. 8-336387P. pastorisMan>8GlcNAc2OCH1 (disruption)Man>8GlcNAc2Contreras et al.WO 02/00856 A2
Japanese Patent Application Publication No. 8-336387 discloses the deletion of an OCH1 homolog in Pichia pastoris. In S. cerevisiae, OCH1 encodes a 1,6-mannosyltransferase, which adds a mannose to the glycan structure Man8GlcNAc2 to yield Man9GlcNAc2. The Man9GlcNAc2 structure, which contains three 1,6 mannose residues, is then a substrate for further 1,2-, 1,6-, and 1,3-mannosyltransferases in vivo, leading to the hypermannosylated glycoproteins that are characteristic for S. cerevisiae and which typically may have 30-40 mannose residues per N-glycan. Because the Och1p initiates the transfer of 1,6 mannose to the Man8GlcNAc2 core, it is often referred to as the “initiating 1,6 mannosyltransferase” to distinguish it from other 1,6 mannosyltransferases acting later in the Golgi. In an och1 mnn1 mnn4 mutant strain of S. cerevisiae, proteins glycosylated with Man8GlcNAc2 accumulate and hypermannosylation does not occur. However, Man8GlcNAc2 is not a substrate for mammalian glycosyltransferases, such as human UDP-GlcNAc transferase I, and accordingly, the use of that mutant strain, in itself, is not useful for producing mammalian-like proteins, i.e., those with complex or hybrid glycosylation patterns.
One can trim Man8GlcNAc2 structures to a Man5GlcNAc2 isomer in S. cerevisiae (although high efficiency trimming greater than 50% in vivo has yet to be demonstrated) by engineering a fungal mannosidase from A. saitoi into the endoplasmic reticulum (ER). The shortcomings of this approach are two-fold: (1) it is not clear whether the Man5GlcNAc2 structures formed are in fact formed in vivo (rather than having been secreted and further modified by mannosidases outside the cell); and (2) it is not clear whether any Man5GlcNAc2 structures formed, if in fact formed in vivo, are the correct isoform to be a productive substrate for subsequent N-glycan modification by GlcNAc transferase I (Maras et al. (1997) Eur. J. Biochem. 249:701-707).
With the objective of providing a more human-like glycoprotein derived from a fungal host, U.S. Pat. No. 5,834,251 discloses a method for producing a hybrid glycoprotein derived from Trichoderma reseei. A hybrid N-glycan has only mannose residues on the Manα1-6 arm of the core mannose structure and one or two complex antennae on the Manα1-3 arm. While this structure has utility, the method has the disadvantage that numerous enzymatic steps must be performed in vitro, which is costly and time-consuming. Isolated enzymes are expensive to prepare and need costly substrates (e.g., UDP-GlcNAc). The method also does not allow for the production of complex glycans on a desired protein.
Intracellular Mannosidase Activity Involved in N-Glycan Trimming
Alpha-1,2-mannosidase activity is required for the trimming of Man8GlcNAc2 to form Man5GlcNAc2, which is a major intermediate for complex N-glycan formation in mammals. Previous work has shown that truncated murine, fungal and human α-1,2-mannosidase cap be expressed in the methylotropic yeast P. pastoris and display Man8GlcNAc2 to Man5GlcNAc2 trimming activity (Lal et al. (1998) Glycobiology 8(10):981-95; Tremblay et al. (1998) Glycobiology 8(6):585-95, Callewaert et al. (2001) FEBS Lett. 503(2-3):173-8). However, to date, no reports exist that show the high level in vivo trimming of Man8GlcNAc2 to Man5GlcNAc2 on a secreted glycoprotein from P. pastoris. 
Moreover, the mere presence of an α-1,2-mannosidase in the cell does not, by itself, ensure proper intracellular trimming of Man8GlcNAc2 to Man5GlcNAc2. (See, e.g., Contreras et al. WO 02/00856 A2, in which an HDEL tagged mannosidase of T. reesei is localized primarily in the ER and co-expressed with an influenza haemagglutinin (HA) reporter protein on which virtually no Man5GlcNAc2 could be detected. See also Chiba et al. (1998) J. Biol. Chem. 273(41): 26298-26304, in which a chimeric α-1,2-mannosidase/Och1p transmembrane domain fusion localized in the ER, early Golgi and cytosol of S. cerevisiae, had no mannosidase trimming activity). Accordingly, mere localization of a mannosidase in the ER or Golgi is insufficient to ensure activity of the respective enzyme in that targeted organelle. (See also, Martinet et al. (1998) Biotech. Letters 20(12): 1171-1177, showing that α-1,2-mannosidase from T. reesei, while localizing intracellularly, increased rather than decreased the extent of mannosylation). To date, there is no report that demonstrates the intracellular localization of an active heterologous α-1,2-mannosidase in either yeast or fungi using a transmembrane localization sequence.
While it is useful to engineer strains that are able to produce Man5GlcNAc2 as the primary N-glycan structure, any attempt to further modify these high mannose precursor structures to more closely resemble human glycans requires additional in vivo or in vitro steps. Methods to further humanize glycans from fungal and yeast sources in vitro are described in U.S. Pat. No. 5,834,251 (supra). If Man5GlcNAc2 is to be further humanized in vivo, one has to ensure that the generated Man5GlcNAc2 structures are, in fact, generated intracellularly and not the product of mannosidase activity in the medium. Complex N-glycan formation in yeast or fungi will require high levels of Man5GlcNAc2 to be generated within the cell because only intracellular Man5GlcNAc2 glycans can be further processed to hybrid and complex N-glycans in vivo. In addition, one has to demonstrate that the majority of Man5GlcNAc2 structures generated are in fact a substrate for GnTI and thus allow the formation of hybrid and complex N-glycans.
Accordingly, the need exists for methods to produce glycoproteins characterized by a high intracellular Man5GlcNAc2 content which can be further processed into human-like glycoprotein structures in non-human eukaryotic host cells, and particularly in yeast and filamentous fungi.
N-Acetylglucosaminyltransferases
N-Acetylglucosaminyltransferases (“GnTs”) belong to another class of glycosylation enzymes that modify N-linked oligosaccharides in the secretory pathway. Such glycosyltransferases catalyze the transfer of a monosaccharide from specific sugar nucleotide donors onto particular hydroxyl position of a monosaccharide in a growing glycan chain in one of two possible anomeric linkages (either α or β). Dennis et al. (1999) Bioessays 21(5):412-21. Specific GnTs add N-acetylglucosamine (“GlcNAc”) onto the Manα1,6 arm or the Manα1,3 arm of an N-glycan substrate (e.g., Man5GlcNAc2 (“mannose-5 core”) and Man3GlcNAc2 (an “inner core structure”)). The reaction product (e.g., GlcNAcMan5GlcNAc2 or GlcNAc2Man3GlcNAc2) can then be modified into bi-, tri-, tetra- and penta-antennary N-linked oligosaccharide structures.
N-Acetylglucosaminyltransferase III (“GnTIII”) is an enzyme that catalyzes the addition of a GlcNAc, on the middle mannose of the trimannose core (Manα1,6 (Manα1,3) Man β1,4-GlcNAc β1,4-GlcNAc β1,4-Asn) of an N-linked oligosaccharide. The addition by GnTIII of a bisecting GlcNAc to an acceptor substrate (e.g. trimannose core) yields a so-called bisected N-glycan. For example, the addition by GnTIII of a bisecting GlcNAc to the GlcNAcMan3GlcNAc2 structure may yield a bisected N-glycan, GlcNAc2Man3GlcNAc2. Similarly, the addition by GnTIII of a bisecting GlcNAc to a GlcNAc2Man3GlcNAc2 structure yields another bisected N-glycan, GlcNAc3Man3GlcNAc2. This latter structure has been implicated in greater antibody-dependent cellular cytotoxicity (ADCC). Umana et al. (1999) Nat. Biotechnol. 17(2):176-80. Other bisected N-glycans can be formed by the action of GnTIII. For example, GlcNAcMan4GlcNAc2 can be converted to bisected GlcNAc2Man4GlcNAc2, Man5GlcNAc2 can be converted to bisected GlcNAcMan5GlcNAc2, and GlcNAcMan5GlcNAc2 can be converted to bisected GlcNAc2Man5GlcNAc2. See, e.g., Narasimhan (1982) J. Biol. Chem. 257:10235-42. Thus far, GnTII activity has only been shown in mammalian cells.
Re-engineering glycoforms of immunoglobulins expressed by mammalian cells is a tedious and cumbersome task. Especially in the case of GnTIII, where over-expression of this enzyme has been implicated in growth inhibition, methods involving regulated (inducible) gene expression had to be employed to produce immunoglobulins with bisected N-glycans. Umana et al. (1999) Biotechnol Bioeng. 65(5):542-9; Umana et al. (1999) Nat. Biotechnol. 17(2):176-80; Umana et al. WO 03/011878; U.S. Pat. No. 6,602,684. Such a growth-inhibition effect complicates the ability to coexpress the target protein and GnTIII and may impose an upper limit on GnTIII overexpression. U.S. Pat. No. 6,602,684. Careful optimization of the expression levels of GnTIII may be necessary. Id. As described above, however, development of the lower eukaryotic host cells used in such a protein production system requires that the endogenous glycosylation pathways of the host cells be further modified.
The enzymes GnTIV, GnTV and GnTIX expressed in mammalian cells are known to catalyze the transfer of GlcNAc residues in particular conformation onto oligosaccharide substrates producing multiantennary glycan structures. UDP-N-acetylglucosamine:α1,3-D-mannoside β1,4N-acetylglucosaminyl-transferase (GnTIV; EC 2.4.1.145) catalyzes the transfer of GlcNAc from UDP-GlcNAc in residues in β1,4 linkage to α1,3-D-mannoside on GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (Gleeson and Schachter, J Biol Chem. 1983 May 25; 258(10):6162-73; Schachter et al., (1989) Methods Enzymol., 179, 351-397). UDP-N-acetylglucosamine:α-6-D-mannoside #1,6-N-acetylglucosaminyltransferase (GnTV; EC 4.1.155) catalyzes the addition of an N-acetylglucosamine to the α1,6 mannosyl core in a β1,6 linkage forming tri- and tetraantennary N-glycans.
Similarly, the expression of GnTVI in avian cells catalyzes the transfer of GlcNAc residues onto oligosaccharide substrates. Specifically, UDP N-acetyl-D glucosamine (GlcNAc):GlcNAcβ1-6(GlcNAc β1-2)Manα1-R[GlcNAc to Man]β1,4-N-acetylglucosaminyltransferase VI(GnTVI) catalyzes the formation of pentaantennary N-glycans (Sakamoto et al., J. Biol. Chem. 2000 Nov. 17; 275(46):36029-34). The gene encoding GnTVI has been purified and isolated recently. Taniguchi et al., JP2002209587A2.
Substrates required to produce complex multiantennary structures have not been synthesized in fungal hosts until recently (Hamilton et al., Science. 2003 Aug. 29; 301 (5637): 1244-6). Mammalian cells typically produce an array of complex glycans such as biantennary, triantennary, tetraantennary and even pentaantennary glycoforms through sequential reaction of specific GnTs. In the Golgi apparatus of such cells, N-glycan processing of glycoproteins produces biantennary structures predominantly, in addition to the formation of triantennary and tetraantennary structures. It is currently understood that in the formation of complex glycans specific GnTs catalyze specific β-GlcNAc linkages (e.g., β1,2; β1,4; β1,6), producing multiantennary glycans in mammalian cells. These cells, however, are incapable of producing any one homogeneous glycoform in high yield.
Recently, lower eukaryotes have been engineered to produce complex glycans in homogeneous forms at significant levels (Science 2003 Aug. 29; 301(5637): 1244-6). The ability to produce multiantennary complex glycans in lower eukaryotes would provide large amounts of properly folded and glycosylated proteins on an industrial scale at low cost, in faster time, safer and in higher quality. What is needed, therefore, is a protein production system utilizing the inherent capability of robust product titers such as those produced in lower eukaryotic host cells (e.g. yeast and filamentous fungi), which is capable of producing multiantennary (and optionally, bisected) N-linked glycans on proteins, especially therapeutic proteins, expressed in these cells.